The present invention is directed to cyclic derivatives containing an imidazole cis amide bond mimetic which bind selectively to somatostatin receptor subtypes. This invention is also directed to methods for making the compounds of the instant invention.
Somatostatin (SRIF) is a cyclic tetradecapeptide hormone containing a disulfide bridge between position 3 and position 14 and has the properties of inhibiting the release of growth hormone (GH) and thyroid-stimulating hormone (TSH), inhibiting the release of insulin and glucagon, and reducing gastric secretion. Metabolism of somatostatin by aminopeptidases and carboxypeptidases leads to a short duration of action.
Somatostatin binds to five distinct receptor (SSTR) subtypes with relatively high affinity for each subtype. The smaller, more rigid analogs of the present invention exhibit high selectivity for several of the receptor subtypes. Binding to the different types of somatostatin subtypes have been associated with the treatment of the following conditions and/or diseases. Activation of types 2 and 5 have been associated with growth hormone suppression and more particularly GH secreting adenomas (Acromegaly) and TSH secreting adenomas. Activation of type 2 but not type 5 has been associated with treating prolactin secreting adenomas. Other indications associated with activation of the somatostatin subtypes are restenosis, inhibition of insulin and/or glucagon and more particularly diabetes mellitus, hyperlipidemia, insulin insensitivity, Syndrome X, angiopathy, proliferative retinopathy, dawn phenomenon and Nephropathy; inhibition of gastric acid secretion and more particularly peptic ulcers, enterocutaneous and pancreaticocutanieous fistula, irritable bowel syndrome, Dumping syndrome, watery diarrhea syndrome, AIDS related diarrhea, chemotherapy-induced diarrhea, acute or chronic pancreatitis and gastrointestinal hormone secreting tumors; treatment of cancer such as hepatoma; inhibition of angiogenesis, treatment of inflammatory disorders such as arthritis; chronic allograft rejection; angioplasty; preventing graft vessel and gastrointestinal bleeding. Somatostatin agonists can also be used for decreasing body weight in a patient.
Somatostatin agonists have also been disclosed to be useful for inhibiting the proliferation of helicobacter pylori. 
In one aspect, the present invention provides a compound of the formula (I), 
or a pharmaceutically acceptable salt thereof,
wherein,
Y and Z for each occurrence are each independently a D- or L-natural or unnatural xcex1-amino acid;
n for each occurrence is independently 0 to 50, provided that both n cannot be 0 at the same time;
m is 0 or an integer from 1 to 10;
a is H or R1;
b is OH, xe2x80x94OR1 or xe2x80x94NR9R9;
or a is taken together with b to form an amide bond;
R1 is independently H, (C1-C4)alkyl or aryl-(C1-C4)alkyl;
R2 is H or an optionally substituted moiety selected from the group consisting of (C1-C4)alkyl, phenyl, phenyl-(C1-C4)alkyl and heterocyclyl-(C1-C4)alkyl, where the optionally substituted moiety is optionally substituted by one or more substituents each independently selected from the group consisting of (C1-C4)alkyl, (C3-C8)cycloalkyl, xe2x80x94Oxe2x80x94R6, xe2x80x94S(O)qxe2x80x94R7, xe2x80x94N(R9R9), xe2x80x94NHCOxe2x80x94R6, xe2x80x94NHSO2R9, xe2x80x94CO2R9, xe2x80x94CONR9R9 and xe2x80x94SO2NR9R9, where q is 0, 1, 2 or 3;
R3 and R4 are each independently H, halo or an optionally substituted moiety selected from the group consisting of (C1-C4)alkyl, (C3-C8)cycloalkyl, aryl and aryl-(C1-C4)alkyl; where the optionally substituted moiety is optionally substituted by one or more substituents selected from the group consisting of OH, (C1-C4)alkyl, (C1-C4)alkoxy, aryloxy, aryl-(C1-C4)alkoxy, xe2x80x94NR9R9, COOH, xe2x80x94CONR9R9 and halo;
or R3 and R4 are taken together with the carbons to which they are attached to form optionally substituted aryl, where the aryl is optionally substituted by one or more substituents each independently selected from the group consisting of OH, (C1-C4)alkyl, (C1-C4)alkoxy, aryloxy, aryl-(C1-C4)alkoxy, xe2x80x94NR9R9, COOR5, xe2x80x94CONR9R9 and halo;
R5 for each occurrence is independently H, or an optionally substituted moiety selected from the group consisting of (C1-C4)alkyl and aryl-(C1-C4)alkyl, where the optionally substituted moiety is optionally substituted by one or more substituents each independently selected from the group consisting of (C1-C4)alkyl, OH, (C1-C4)alkoxy, aryloxy, NO2, aryl-(C1-C4,)alkoxy, xe2x80x94NR9R9, COOH, xe2x80x94CONR9R9 and halo;
R6 for each occurrence is independently selected from the group consisting of H, (C1-C4)alkyl, (C1-C4)alkoxy, aryl-(C1-C4)alkyl and aryl-(C1-C4)alkoxy;
R7 is H when q is 3 or, R7 for each occurrence is independently selected from the group consisting of (C1-C4)alkyl, aryl and aryl-(C1-C4)alkyl when q is 0, 1 or 2; and
R9 for each occurrence is independently selected from the group consisting of H, NO2, (C1-C4)alkyl, aryl and aryl-(C1-C4)alkyl.
A preferred compound of formula (I) is the compound H-Trp-D-Trp-Lys-Abu-Phe xcexa8 (4-(3-methoxyphenyl)imidazole)-Gly-OH.
In another aspect, the present invention provides a compound of the formula (II), 
or a pharmaceutically acceptable salt thereof,
wherein,
Y and Z for each occurrence are each independently a D- or L-natural or unnatural xcex1-amino acid;
m is 0 or an integer from 1 to 10;
n for each occurrence is independently 0 to 6;
R1 for each occurrence is independently H, (C1-C4)alkyl or aryl-(C1-C4)alkyl;
R2 is H or an optionally substituted moiety selected from the group consisting of (C1-C4)alkyl, phenyl, phenyl-(C1-C4)alkyl and heterocyclyl-(C1-C4)alkyl, where the optionally substituted moiety is optionally substituted by one or more substituents each independently selected from the group consisting of (C1-C4)alkyl, cycloalkyl, xe2x80x94Oxe2x80x94R6, xe2x80x94S(O)qxe2x80x94R7, xe2x80x94N(R9R9), xe2x80x94NHCOxe2x80x94R8, xe2x80x94NHSO2R9, xe2x80x94CO2R9, xe2x80x94CONR9R9 and xe2x80x94SO2NR9R9, where q is b, 1, 2 or 3;
R3 and R4 are each independently H, halo or an optionally substituted moiety selected from the group consisting of (C1-C4)alkyl, cycloalkyl, aryl and aryl-(C1-C4)alkyl; where the optionally substituted moiety is optionally substituted by one or more substituents selected from, the group consisting of OH, (C1-C4)alkyl, (C1-C4)alkoxy, aryloxy, aryl-(C1-C4)alkoxy, xe2x80x94NR9R9, COOH, xe2x80x94CONR9R9 and halo;
or R3 and R4 are taken together with the carbons to which they are attached to form optionally substituted aryl, where the aryl is optionally substituted by one or more substituents each independently selected from the group consisting of OH, (C1-C4)alkyl, (C1-C4)alkoxy, aryloxy, aryl-(C1-C4)alkoxy, xe2x80x94NR9R9, COOR5, xe2x80x94CONR9R9 and halo;
R5 for each occurrence is independently H, or an optionally substituted moiety selected from the group consisting of (C1-C4)alkyl and aryl-(C1-C4)alkyl, where the optionally substituted moiety is optionally substituted by one or more substituents each independently selected from the group consisting of (C1-C4)alkyl, OH, (C1-C4)alkoxy, aryloxy, NO2, aryl-(C1-C4)alkoxy, xe2x80x94NR9R9, COOH, xe2x80x94CONR9R9 and halo;
R6 for each occurrence is independently selected from the group consisting of H, (C1-C4)alkyl, (C1-C4)alkoxy, aryl-(C1-C4)alkyl and aryl-(C1-C4)alkoxy;
R7 is H when q is 3, or R7 for each occurrence is independently selected from the group consisting of (C1-C4)alkyl, aryl and aryl-(C1-C4)alkyl when q is 0, 1 or 2; and
R9 for each occurrence is independently selected from the group consisting of H, NO2, (C1-C4)alkyl, aryl and aryl-(C1-C4)alkyl.
X1 is a natural or unnatural D- or L-xcex1-amino acid, where when X1 is Phe, NaI, Trp, Tyr, PaI or His the aromatic ring thereof is optionally substituted on carbon or nitrogen by R6 or when X1 is Ser or Thr, the side chain oxygen is optionally substituted by one or more R1;
X2 is D- or L-Trp, N-methyl-D-Trp or N-methyl-L-Trp;
X3 is Lys, xcex1-N-methyl-Lys or xcex5-N-(C1-C4)alkyl-Lys or xcex5-N-[aryl-(C1-C4)alkyl]-Lys;
X4 is a natural or unnatural D- or L-xcex1-amino acid where when X4 is Phe, NaI, Trp, Tyr or His, the aromatic ring thereof is optionally substituted on carbon or nitrogen by R8 or when X4 is Ser, Tyr or Thr, the side chain oxygen may be substituted with one or more R1.
The bonds between X1, X2, X3 and X4 are amide bonds as is the bond between X1 and Z, and the bond between X4 and Y.
A preferred group of compounds of formula (II), designated group A, is wherein,
each n is 2;
m is 0 or 1 to 5;
R1 for each occurrence is independently H, methyl or aryl-(C1-C4)alkyl;
R2 is an optionally substituted moiety selected from the group consisting phenyl-(C1-C4)alkyl and heterocyclyl-(C1-C4)alkyl, where the optionally substituted moiety is substituted by a substituent selected from the group consisting of (C1-C4)alkyl and xe2x80x94Oxe2x80x94R6; and
R3 and R4 are each independently H, halo or an optionally substituted moiety selected from the group consisting of (C1-C4)alkyl and aryl; where the optionally substituted moiety is optionally substituted by a substituent selected from the group consisting of OH, (C1-C4)alkoxy, aryloxy and halo.
A preferred group of the.group A compounds, designated group B, is wherein
X1 is Phe, NaI, Trp, Tyr, PaI or His, wherein the aromatic ring thereof is optionally substituted on carbon or nitrogen by R6; and
X4 is Val, Abu, Ser, Thr, NaI, Trp, Tyr or His, wherein the aromatic ring of NaI, Trp, Tyr and His is optionally substituted on carbon and/or nitrogen by R8 or when X4 is Ser, Tyr or Thr, the side chain oxygen is optionally substituted by R1.
A preferred group of the group B compounds, designated group C, is wherein
X1 is Phe, Trp or Tyr wherein the aromatic ring thereof is optionally substituted on carbon or nitrogen by R6;
X2 is D-Trp or N-methyl-D-Trp;
X4 is Val, Thr, Abu, NaI or Tyr, wherein the side chain oxygen of the hydroxy group of Thr and Tyr is optionally substituted by R1;
R1 for each occurrence is independently H, methyl or benzyl;
R2 is an optionally substituted moiety selected from the group consisting phenylmethyl and heterocyclyl-methyl, where the optionally substituted moiety is substituted by a substituent selected from the group consisting of (C1-C4)alkyl and xe2x80x94Oxe2x80x94R6;
R3 is (C1-C4)alkyl or optionally substituted aryl; where the optionally substituted aryl is substituted by a substituent selected from the group consisting of OH, (C1-C4)alkoxy, aryloxy, and halo;
R4 is H; and
R6 for each occurrence is independently selected from the group consisting of H and aryl-(C1-C4)alkoxy.
A preferred group of the group C compounds, designated group D, is wherein X1 is Phe, Trp, Tyr or Tyr(OBzl);
X4 is Val, Thr, Abu, NaI, or Tyr, wherein the hydroxy group of Thr and Tyr is optionally substituted benzyl;
m is 0, 2 or 4;
R2 is an optionally substituted moiety selected from the group consisting of phenylmethyl and 3-indolylmethyl where the optionally substituted moiety is optionally substituted by xe2x80x94Oxe2x80x94R6; and
R3 is 1,1-dimethylethyl or optionally substituted aryl; where the optionally substituted aryl is optionally substituted by a moiety selected from the group consisting of OH, (C1-C4)alkoxy and halo.
A preferred group of the group D compounds, designated group E, is wherein
R2 is phenylmethyl;
R3 is 1,1-dimethylethyl or optionally substituted phenyl, where the optionally substituted phenyl is optionally substituted by OH or OCH3; and
R6 for each occurrence is independently selected from the group consisting of H and benzylmethoxy.
A preferred group of the group, E compounds, designated group F, is
cyclo[Tyr-D-Trp-Lys-Val-Phexcexa8(4-(3-methoxyphenyl)imidazole)-Gly],
cyclo[Tyr(OBzl)-D-Trp-Lys-Val-Phexcexa8(4-(3-methoxyphenyl)imidazole)-Gly],
cyclo[Trp-D-Trp-Lys-Val-Phexcexa8(4-(3-methoxyphenyl)imidazole)-Gly],
cyclo[Trp-D-Trp-Lys-Val-Phexcexa8(4-(3-hydroxyphenyl)imidazole)-Gly],
cyclo[Trp-D-Trp-Lys-Thr(OBzl)-Phexcexa8(4-(3-methoxyphenyl)imidazole)-Gly],
cyclo[Trp-D-Trp-Lys-Thr-Phexcexa8(4-(3-hydroxyphenyl)imidazole)-Gly],
cyclo[Trp-D-Trp-Lys-Abu-Phexcexa8(4-(3-methoxyphenyl)imidazole)-Gly],
cyclo[Phe-D-Trp-Lys-Tyr(OBzl)-Phexcexa8(4-(1,1-dimethylethyl)imidazole)-Gly],
cyclo[Phe-D-Trp-Lys-Val-Phexcexa8(4-(3-methoxyphenyl)imidazole)-Gly],
cyclo[Phe-D-Trp-Lys-Tyr(OBzl)-Phexcexa8(4-(3-methoxyphenyl)imidazole)-Gly],
cyclo[Phe-D-Trp-Lys-Tyr-Phexcexa8(4-(3-methoxyphenyl)imidazole)-Gly],
cyclo[Phe-D-Trp-Lys-Tyr-Phexcexa8(4-(3-hydroxyphenyl)imidazole)-Gly],
cyclo[Trp-D-Trp-Lys-Tyr(Bzl)-Phexcexa8(4-(3-methoxyphenyl)imidazole)-Gly],
cyclo[Tyr-D-Trp-Lys-Val-Phexcexa8(4-(3-hydroxyphenyl)imidazole)-Gly],
cyclo[Phe-D-Trp-Lys-NaI-Phexcexa8(4-(3-hydroxyphenyl)imidazole)-Gly],
cyclo[Phe-D-Trp-Lys-NaI-Phexcexa8(4-(3-methoxyphenyl)imidazole)-Gly],
cyclo[Trp-D-Trp-Lys-Tyr(OBzl)-Phexcexa8(4-(3-methoxyphenyl)imidazole)-(xcex3)Abu],
cyclo[Trp-D-Trp-Lys-Tyr(OBzl)-Phexcexa8(4-(4-methoxyphenyl)imidazole)-Gly],
cyclo[Trp-D-Trp-Lys-Tyr(OBzl)-Phexcexa8(4-(phenyl)imidazole)-Gly],
cyclo[Trp-D-Trp-Lys-Tyr(OBzl)-Phexcexa8(4-(3-methoxyphenyl)imidazole)-(xcex5)Ahx] and
cyclo[Trp-D-Trp-Lys-Tyr(OBzl)-Phexcexa8(4-(3-hydroxyphenyl)imidazole)-(xcex3)Abu].
A preferred group of the group F compounds, designated group G, is
cyclo[Tyr-D-Trp-Lys-Val-Phexcexa8(4-(3-methoxyphenyl)imidazole)-Gly],
cyclo[Tyr(OBzl)-D-Trp-Lys-Val-Phexcexa8(4-(3-methoxyphenyl)imidazole)-Gly],
cyclo[Trp-D-Trp-Lys-Val-Phexcexa8(4-(3-methoxyphenyl)imidazole)-Gly],
cyclo[Trp-D-Trp-Lys-Val-Phexcexa8(4-(3-hydroxyphenyl)imidazole)-Gly],
cyclo[Trp-D-Trp-Lys-Thr(OBzl)-Phexcexa8(4-(3-methoxyphenyl)imidazole)-Gly],
cyclo[Trp-D-Trp-Lys-Thr-Phexcexa8(4-(3-hydroxyphenyl)imidazole)-Gly],
cyclo[Trp-D-Trp-Lys-Abu-Phexcexa8(4-(3-methoxyphenyl)imidazole)-Gly],
cyclo[Phe-D-Trp-Lys-Tyr(OBzl)-Phexcexa8(4-(1,1-dimethylethyl)imidazole)-Gly],
cyclo[Phe-D-Trp-Lys-Val-Phexcexa8(4-(3-methoxyphenyl)imidazole)-Gly],
cyclo[Phe-D-Trp-Lys-Tyr-Phexcexa8(4-(3-hydroxyphenyl)imidazole)-Gly] and
cyclo[Tyr-D-Trp-Lys-Val-Phexcexa8(4-(3-hydroxyphenyl)imidazole)-Gly].
A preferred group of the group G compounds, designated group H, is
cyclo[Tyr-D-Trp-Lys-Val-Phexcexa8(4-(3-methoxyphenyl)imidazole)-Gly],
cyclo[Trp-D-Trp-Lys-Val-Phexcexa8(4-(3-methoxyphenyl)imidazole)-Gly],
cyclo[Trp-D-Trp-Lys-Val-Phexcexa8(4-(3-hydroxyphenyl)imidazole)-Gly],
cyclo[Trp-D-Trp-Lys-Thr-Phexcexa8(4-(3-hydroxyphenyl)imidazole)-Gly],
cyclo[Trp-D-Trp-Lys-Abu-Phexcexa8(4-(3-methoxyphenyl)imidazole)-Gly],
cyclo[Phe-D-Trp-Lys-Val-Phexcexa8(4-(3-methoxyphenyl)imidazole)-Gly] or
cyclo[Tyr-D-Trp-Lys-Val-Phexcexa8(4-(3-hydroxyphenyl)imidazole)-Gly].
A preferred group of the group H compounds, designated group I, is
cyclo[Tyr-D-Trp-Lys-Val-Phexcexa8(4-(3-methoxyphenyl)imidazole)-Gly],
cyclo[Trp-D-Trp-Lys-Val-Phexcexa8(4-(3-hydroxyphenyl)imidazole)-Gly],
cyclo[Trp-D-Trp-Lys-Thr-Phexcexa8(4-(3-hydroxyphenyl)imidazole)-Gly] and
cyclo[Tyr-D-Trp-Lys-Val-Phexcexa8(4-(3-hydroxyphenyl)imidazole)-Gly].
Another preferred group of the group F compounds, designated group J, is
cyclo[Tyr-D-Trp-Lys-Val-Phexcexa8(4-(3-methoxyphenyl)imidazole)-Gly],
cyclo[Trp-D-Trp-Lys-Val-Phexcexa8(4-(3-methoxyphenyl)imidazole)-Gly],
cyclo[Trp-D-Trp-Lys-Val-Phexcexa8(4-(3-hydroxyphenyl)imidazole)-Gly],
cyclo[Trp-D-Trp-Lys-Thr(OBzl)-Phexcexa8(4-(3-methoxyphenyl)imidazole)-Gly],
cyclo[Trp-D-Trp-Lys-Thr-Phexcexa8(4-(3-hydroxyphenyl)imidazole)-Gly],
cyclo[Trp-D-Trp-Lys-Abu-Phexcexa8(4-(3-methoxyphenyl)imidazole)-Gly],
cyclo[Phe-D-Trp-Lys-Tyr(OBzl)-Phexcexa8(4-(1,1-dimethylethyl)imidazole)-Gly],
cyclo[Phe-D-Trp-Lys-Tyr(OBzl)-Phexcexa8(4-(3-methoxyphenyl)imidazole)-Gly],
cyclo[Phe-D-Trp-Lys-Tyr-Phexcexa8(4-(3-hydroxyphenyl)imidazole)-Gly],
cyclo[Trp-D-Trp-Lys-Tyr(OBzl)-Phexcexa8(4-(3-methoxyphenyl)imidazole)-Gly],
cyclo[Tyr-D-Trp-Lys-Val-Phexcexa8(4-(3-hydroxyphenyl)imidazole)-Gly],
cyclo[Phe-D-Trp-Lys-NaI-Phexcexa8(4-(3-hydroxyphenyl)imidazole)-Gly],
cyclo[Phe-D-Trp-Lys-NaI-Phexcexa8(4-(3-methoxyphenyl)imidazole)-Gly],
cyclo[Trp-D-Trp-Lys-Tyr(OBzl)-Phexcexa8(4-(3-methoxyphenyl)imidazole)-(xcex3)Abu],
cyclo[Trp-D-Trp-Lys-Tyr(OBzl)-Phexcexa8(4-(4-methoxyphenyl)imidazole)-Gly],
cyclo[Trp-D-Trp-Lys-Tyr(OBzl)-Phexcexa8(4-(phenyl)imidazole)-Gly],
cyclo[Trp-D-Trp-Lys-Tyr(OBzl)-Phexcexa8(4-(3-methoxyphenyl)imidazole)-(xcex5)Ahx] and
cyclo[Trp-D-Trp-Lys-Tyr(OBzl)-Phexcexa8(4-(3-hydroxyphenyl)imidazole)-(xcex3)Abu].
A preferred group of the group.J compounds, designated group K, is
cyclo[Trp-D-Trp-Lys-Thr-Phexcexa8(4-(3-hydroxyphenyl)imidazole)-Gly],
cyclo[Trp-D-Trp-Lys-Tyr(OBzl)-Phexcexa8(4-(3-methoxyphenyl)imidazole)-Gly],
cyclo[Phe-D-Trp-Lys-NaI-Phexcexa8(4-(3-hydroxyphenyl)imidazole)-Gly],
cyclo[Trp-D-Trp-Lys-Tyr(OBzl)-Phexcexa8(4-(3-methoxyphenyl)imidazole)-(xcex3)Abu],
cyclo[Trp-D-Trp-Lys-Tyr(OBzl)-Phexcexa8(4-(4-methoxyphenyl)imidazole)-Gly] or
cyclo[Trp-D-Trp-Lys-Tyr(OBzl)-Phexcexa8(4-(phenyl)imidazole)-Gly].
A preferred group of the group K compounds, designated group L, is
cyclo[Trp-D-Trp-Lys-Tyr(OBzl)-Phexcexa8(4-(3-methoxyphenyl)imidazole)-Gly],
cyclo[Trp-D-Trp-Lys-Tyr(OBzl)-Phexcexa8(4-(3-methoxyphenyl)imidazole)-(xcex3)Abu],
cyclo[Trp-D-Trp-Lys-Tyr(OBzl)-Phexcexa8(4-(4-methoxyphenyl)imidazole)-Gly] and
cyclo[Trp-D-Trp-Lys-Tyr(OBzl)-Phexcexa8(4-(phenyl)imidazole)-Gly].
In another aspect, the present invention provides a pharmaceutical composition comprising an effective amount of a compound of formula (I) or formula (II) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
In still another aspect, the present invention provides a method of eliciting a somatostatin receptor agonist effect in a mammal in need thereof, which comprises administering to said mammal an effective amount of a compound of formula (I) or formula (II) or a pharmaceutically acceptable salt thereof.
In yet another aspect, the present invention provides a method of eliciting a somatostatin receptor antagonist effect in a mammal in need thereof, which comprises administering to said mammal an effective amount of a compound according of formula (I) or formula (II) or a pharmaceutically acceptable salt thereof.
In another aspect, the present invention provides a method of treating prolactin secreting adenomas, restenosis, diabetes mellitus, hyperlipidemia, insulin insensitivity, Syndrome X, angiopathy, proliferative retinopathy, dawn phenomenon, Nephropathy, gastric acid secretion, peptic ulcers, enterocutaneous and pancreaticocutaneous fistula, irritable bowel syndrome, Dumping syndrome, watery diarrhea syndrome, AIDS related diarrhea, chemotherapy-induced diarrhea, acute or chronic pancreatitis, gastrointestinal hormone secreting tumors, cancer, hepatoma, angiogenesis, inflammatory disorders, arthritis, chronic allograft rejection, angioplasty, graft vessel bleeding or gastrointestinal bleeding, in a mammal in need thereof, which comprises administering to said mammal a compound of formula (I) or formula (II) or a pharmaceutically acceptable salt thereof.
In another aspect, this invention provides a method of inhibiting the proliferation of helicobacter pylori in a mammal in need thereof, which comprises administering to said mammal a compound of formula (I) or formula (II) or a pharmaceutically acceptable salt thereof.
In another aspect, this invention provides a process for preparing a compound of the formula 
which-comprises deprotecting a compound of the formula 
by cleaving the Prt group;
wherein,
Prt is an amino acid side chain protecting group;
Y and Z are each independently a D- or L-natural or unnatural xcex1-amino acid optionally having a protected side chain, where the Hxe2x80x94N* is the amino group of the N-terminal amino acid defined by Y and Oxe2x95x90C* is the carboxyl group of the C-terminal amino acid defined by Z;
n for each occurrence is independently 1 to 50;
and all other variables are as defined for formula (I) shown hereinabove.
In another aspect, the present invention provides, a process for preparing a compound of the formula 
which comprises: 
for a compound of formula (a), forming an amide bond between the terminal amino group of the last amino acid defined by Y and the terminal carboxyl group of the last amino acid defined by Z by reacting a compound of the formula (axe2x80x2) with a peptide coupling reagent and an additive; or
for a compound of formula (b), forming an amide bond between the terminal amino group, and the terminal carboxyl group of the last amino acid defined by Z by reacting a compound of the formula (bxe2x80x2) with a peptide coupling reagent and an additive; or
for a compound of formula (c), forming an amide bond between the terminal amino group of the last amino acid defined by Y and the terminal carboxyl group by reacting a compound of the formula (cxe2x80x2) with a peptide coupling reagent and an additive;
wherein, Prt is an amino acid side chain protecting group;
Y and Z are each independently a D- or L-natural or unnatural xcex1-amino acid optionally having a protected side chain, where the Hxe2x80x94N* is the amino group of the N-terminal amino acid defined by Y and Oxe2x95x90C* is the carboxyl group of the C-terminal amino acid defined by Z;
n for each occurrence is independently 1 to 50;
and all other variables are as defined for formula (I) shown hereinabove.
In still another aspect, the present invention provides a process for preparing a compound of the formula 
which comprises reacting a compound of the formula 
with an xcex1-halo ketone of the formula Xxe2x80x2xe2x80x94CH(R3)CO(R4) in the presence of a base and a polar aprotic solvent until the reaction is substantially complete; evaporating the polar aprotic solvent to yield a solid; dissolving the solid in an aprotic organic solvent and an excess amount of aqueous NH4OAc to form a solution; and refluxing the solution and concurrently removing that polar layer to yield a compound of formula (A); wherein
X is an amine protecting group;
Xxe2x80x2 is halo;
and all other variables are as defined for formula (I) shown hereinabove.
In yet another aspect, this invention provides a process for preparing a compound of the formula (I), 
which comprises coupling a compound of the formula (B), 
with an Nxcex1-protected amino acid, (Prt)-Y, where the Nxcex1-protected amino acid is in the form of its activated ester, anhydride or acid halide, in the presence of a base until the reaction is substantially complete to yield a compound of the formula (C), 
xe2x80x83optionally deprotecting the amino group of the Nxcex1-protected amino acid, (Prt)-Y, using a conventional deprotecting reaction and repeating the coupling reaction with another Nxcex1-protected amino acid repeatedly until the desired compound of formula (I) is obtained;
Y for each occurrence is independently a D- or L-natural or unnatural xcex1-amino acid optionally having a side chain with a protecting group;
Prt is an amine protecting group;
Rxe2x80x2 is an alkyl ester or benzyl ester;
n is 1 to 100;
and all other variables are as defined for formula (I) shown hereinabove.
In another aspect, this invention provides a process for preparing a compound of formula (I), as defined hereinabove, which comprises coupling a compound of formula (B), activated as it""s active ester, anhydride, or acid halide, with an N-deprotected peptide-resin (Axe2x80x2), prepared by methods well known to those familiar with the peptide synthesis, deprotecting the N-terminal Fmoc group using piperidine in DMF, TAEA, or similar base and deprotecting and cleaving the resulting intermediate (Bxe2x80x2) from the resin using a strong acid. All variables are as defined for formula (I) shown hereinabove. 
In another aspect, this invention provides, a process for preparing a compound of formula (I), which comprises coupling a compound of formula (B), activated as it""s active ester, anhydride, or acid halide, with an N-deprotected peptide-resin (H), prepared by methods well known to those familiar with peptide synthesis, deprotecting the N-terminal Fmoc group using piperidine in DMF, TAEA, or similar base, acylating the liberated N-terminal amino group with an Nxcex1-Fmoc-protected amino acid (x) using peptide coupling reactions well known to those familiar with the art, repeating the base deprotection and coupling steps as required to incorporate additional amino acids (x), deprotecting and cleaving the resulting intermediate (Cxe2x80x2) from the resin using a strong acid. All variables are as defined for formula (I) shown hereinabove. 
In another aspect, this invention provides, a process for preparing a compound of formula (I), which comprises coupling a compound of formula (B), activated as it""s active ester, anhydride, or acid halide, to an amino-substituted resin, such as Tris-(alkoxy)-benzylamine resin (PAL Resin), 4-(2xe2x80x2,4xe2x80x2-Dimethoxyphenyl-aminomethyl)-phenoxy Resin (N-deprotected Rink resin), or Benzhydrylamine resin, deprotecting the N-terminal terminal Fmoc group using piperidine in DMF, TAEA, or similar base, acylating the liberated N-terminal amino group with an Nxcex1-Fmoc-protected amino acid (x) using peptide coupling reactions well known to those familiar with the art, repeating the base deprotection and coupling steps as required to incorporate additional amino acids (x), deprotecting and cleaving the resulting intermediate (Dxe2x80x2) from the resin using a strong acid and all other variables are as defined for formula (I) shown hereinabove. All variables are as defined for formula (I) shown hereinabove. 
In another aspect, this invention provides a process for preparing a compound of formula (I), which comprises reacting a compound of formula (B) with a base, such as Cs2CO3, reacting the resulting phenolic cesium salt (Exe2x80x2) with a halomethylated polystyrene resin, such as Merrifield peptide resin, removing the Fmoc protecting group with piperidine or similar organic base, acylating the liberated N-terminal amino group with an Nxcex1-Fmoc-protected amino acid (x) using peptide coupling reactions well known to those familiar with the art, repeating the base deprotection and coupling steps as required to incorporate additional amino acids (x), deprotecting the final protected peptide sequence at the N-terminus with piperidine or similar organic base and at the C-terminus with Tfa, cyclizing the resulting intermediate (Fxe2x80x2) using peptide coupling reactions well known to those familiar with the art, and cleaving the resulting intermediate (Gxe2x80x2) from the resin using a strong acid. All variables are as defined for formula (I) shown hereinabove. 
In another aspect, this invention provides a process for preparing a compound of formula (I), as defined hereinabove, which comprises coupling a compound of formula (B), activated as it""s active ester, anhydride, or acid halide, with an N-deprotected peptide-resin (Axe2x80x2), prepared by methods well known to those familiar with the art, deprotecting the N-terminal Boc group using Tfa, and deprotecting side chain protecting groups and cleaving the resulting intermediate (Hxe2x80x2) from the resin using a strong acid such as HF. All variables are as defined for formula (I) shown hereinabove. 
In another aspect, this invention provides a process for preparing a compound of formula (I), which comprises coupling a compound of formula (B), activated as it""s active ester, anhydride, or acid halide, with an N-deprotected peptide-resin (H), prepared by methods well known to those familiar with the art, deprotecting the N-terminal Boc group using Tfa, acylating the liberated N-terminal amino group with an Nxcex1-Boc-protected amino acid (x) using peptide coupling reactions well known to those familiar with the art, repeating the Tfa deprotection and coupling steps as required to incorporate additional amino acids (x), deprotecting and cleaving the resulting intermediate (Ixe2x80x2) from the resin using a strong acid. All variables are as defined for formula (I) shown hereinabove. 
In another aspect, this invention provides a process for preparing a compound of formula (I), which comprises reacting a compound of formula (B) with a base, such as Cs2CO3, reacting the resulting phenolic cesium salt (Jxe2x80x2) with a halomethylated polystyrene resin, such as Merrifield peptide resin, removing the Boc protecting group with Tfa, acylating the liberated N-terminal amino group with an Nxcex1-Boc-protected amino acid (x) using peptide coupling reactions well known to those familiar with the art, repeating the Tfa deprotection and coupling steps as required to incorporate additional amino acids (x), deprotecting the final protected peptide sequence at the N-terminus with. Tfa and at the C-terminus with an inorganic base such as LiOH in aqueous DMF, cyclizing the resulting intermediate (Kxe2x80x2) with using peptide coupling reactions well known to those familiar with the art, and cleaving the resulting intermediate (Lxe2x80x2) from the resin using a strong acid. All variables are as defined for formula (I) shown hereinabove. 
In another aspect, this invention provides a process for preparing a compound of formula (I), which comprises coupling a compound of formula (B), activated as it""s active ester, anhydride, or acid halide, with an N-deprotected peptide, 4-Nitrobenzophenone oxime resin (Mxe2x80x2), prepared by methods well known to those familiar with the art, deprotecting the N-terminal Boc group using Tfa, acylating the liberated N-terminal amino group with an Nxcex1-Boc-protected amino acid (x) using peptide coupling reactions well known to those familiar with the art, repeating the Tfa deprotection and coupling steps as required to incorporate additional amino acids (x), deprotecting the N-terminal Boc group with Tfa, cyclizing and cleaving the resulting intermediate Nxe2x80x2-deprotected intermediate (Nxe2x80x2) by neutralizing with a suitable organic base, and removing side chain protecting groups with a strong acid, such as HF. All variables are as defined for formula (I) shown hereinabove. 
The term heterocycle, as used herein, represents any heterocycle that may appear in the side chain of an amino acid. Examples include, but are not limited to, such heterocycles as benzothienyl, coumaryl, imidazolyl, indolyl, purinyl, pyridyl, pyrimidinyl, quinolinyl, thiazolyl, thienyl and triazolyl.
The term aryl as used herein, is intended to mean any stable monocyclic or bicyclic carbon ring of up to 7 members in each ring, wherein at least one ring is aromatic. Examples of aryl groups include biphenyl, indanyl, naphthyl, phenyl, and 1,2,3,4-tetrahydronaphthalene.
In the instant application several abbreviated designations are used for the amino acid components, certain preferred protecting groups, reagents and solvents. The meanings of such abbreviated designations are given in Table 1.
In Vitro Assay
The affinity of a compound for human somatostatin subtype receptors 1 to 5 (sst1, sst2, sst3, sst4 and sst5, respectively) is determined by measuring the inhibition of [125I-Tyr11]SRIF-14 binding to CHO-K1 transfected cells.
The human sst1 receptor gene was cloned as a genomic fragment. A 1.5 Kb PstI-XmnI segment containing 100 bp of the 5xe2x80x2-untranslated region, 1.17 Kb of the entire coding region, and 230 bp of the 3xe2x80x2-untranslated region was modified by the BglII linker addition. The resulting DNA fragment was subcloned into the BamHI site of a pCMV-81 to produce the mammalian expression plasmid (provided by Dr. Graeme Bell, Univ. Chicago). A clonal cell line stably expressing the sst, receptor was obtained by transfection into CHO-K1 cells (ATCC) using the calcium phosphate co-precipitation method (1). The plasmid pRSV-neo (ATCC) was included as a selectable marker. Clonal cell lines were selected in RPMI 1640-media containing 0.5 mg/ml of G418 (Gibco), ring cloned, and expanded into culture.
The human sst2 somatostatin receptor gene, isolated as a 1.7 Kb BamHI-HindIII genomic DNA fragment and subcloned into the plasmid vector pGEM3Z (Promega), was kindly provided by Dr. G. Bell (Univ. of Chicago). The mammalian cell expression vector is constructed by inserting the 1.7 Kb BamHI-HindII fragment into compatible restriction endonuclease sites in the plasmid pCMV5. A clonal cell line is obtained by transfection into CHO-K1 cells using the calcium phosphate co-precipitation method. The plasmid pRSV-neo is included as a selectable marker.
The human sst3 was isolated at genomic fragment, and the complete coding sequence was contained within a 2.4 Kb BamHI/HindIII fragment. The mammalian expression plasmid, pCMV-h3 was constructed by inserting the a 2.0 Kb NcoI-HindIII fragment into the EcoR1 site of the pCMV vector after modification of the ends and addition of EcoR1 linkers. A clonal cell line stably expressing the sst3 receptor was obtained by transfection into CHO-K1 cells (ATCC) using the calcium phosphate co-precipitation method. The plasmid pRSV-neo (ATCC) was induced as a selectable marker. Clonal cell lines were selected in RPMI 1640-media containing 0.5 mg/ml of G418 (Gibco), ring cloned, and expanded into culture.
The human sst4 receptor expression plasmid, pCMV-HX was provided by Dr. Graeme Bell (Univ. Chicago). The vector contains the 1.4 Kb NheI-NheI genomic fragment encoding the human sst4, 456 bp of the 5xe2x80x2-untranslated region and 200 bp of the 3xe2x80x2-untranslated region, clone into the XbaI/EcoR1 sites of PCMV-HX. A clonal cell line stably expressing the sst4 receptor was obtained by transfection into CHO-K1 cells (ATCC) using the calcium phosphate co-precipitation method. The plasmid pRSV-neo (ATCC) was included as a selectable marker. Clonal cell lines were selected in RPMI 1640-media containing 0.5 mg/ml of G418 (Gibco), ring cloned, and expanded into culture.
The human sst5 gene was obtained by PCR using a xcex genomic clone as a template, and kindly provided by Dr. Graeme Bell (Univ. Chicago). The resulting 1.2 Kb PCR fragment contained 21 base pairs of the 5xe2x80x2-untranslated region, the full coding region, and 55 bp of the 3xe2x80x2-untranslated region. The clone was inserted into EcoR1 site of the plasmid pBSSK(+). The insert was recovered as a 1.2 Kb HindIII-XbaI fragment for subcloning into pCVM5 mammalian expression vector. A clonal cell line stably expressing the SST5 receptor was obtained by transfection into CHO-K1 cells (ATCC) using the calcium phosphate co-precipitation method. The plasmid pRSV-neo (ATCC) was included as a selectable marker. Clonal cell lines were selected in RPMI 1640-media containing 0.5 mg/ml of G418 (Gibco), ring cloned, and expanded into culture.
CHO-K1 cells stably expressing one of the human sst receptor are grown in RPMI 1640 containing 10% fetal calf serum and 0.4 mg/ml geneticin. Cells are collected with 0.5 mM EDTA, and centrifuged at 500 g for about 5 min. at about 4xc2x0 C. The pellet is resuspended in 50 mM Tris, pH 7.4 and centrifuged twice at 500 g for about 5 min. at about 4xc2x0 C. The cells are lysed by sonication and centrifuged at 39000 g for about 10 min. at about 4xc2x0 C. The pellet is resuspended in the same buffer and centrifuged at 50000 g for about 10 min. at about 4xc2x0 C. and membranes in resulting pellet are stored at xe2x88x9280xc2x0 C.
Competitive inhibition experiments of [125I-Tyr11]SRIF-14 binding are run in duplicate in polypropylene 96 well plates. Cell membranes (10 xcexcg protein/well) are incubated with [125I-Tyr11]SRIF-14 (0.05 nM) for about 60 min. at about 37xc2x0 C. in 50 mM HEPES (pH 7.4), 0.2% BSA, 5 mM MgCl2, 200 KIU/ml Trasylol, 0.02 mg/ml bacitracin and 0.02 mg/ml phenylmethylsulphonyl fluoride.
Bound from free [125I-Tyr11]SRIF-14 is separated by immediate filtration through GF/C glass fiber filter plate (Unifilter, Packard) presoaked with 0.1% polyethylenimine (P.E.I.), using Filtermate 196 (Packard) cell harvester. Filters are washed with 50 mM HEPES at about 0-4xc2x0 C. for about 4 sec. and assayed for radioactivity using Packard Top Count.
Specific binding is obtained by subtracting nonspecific binding (determined in the presence of 0.1 xcexcM SRIF-14) from total binding. Binding data are analyzed by computer-assisted nonlinear regression analysis (MDL) and inhibition constant (Ki) values are determined.
The determination of whether a compound of the instant invention is an agonist or an antagonist is determined by the following assay.
Functional Assay: Inhibition of cAMP Intracellular Production:
CHO-K1 Cells expressing human somatostatin (SRIF-14) subtype receptors are seeded in 24-well tissue culture multidishes in RPMI 1640-media with 10% FCS and 0.4 mg/ml geneticin. The medium is changed the day before the experiment.
Cells at 105 cells/well are washed 2 times by 0.5 ml and fresh RPMI with 0.2% BSA supplemented with 0.5 mM (1) 3-isobutyl-1-methylxanthine (IBMX) and incubated for about 5 min at about 37xc2x0 C.
Cyclic AMP production is stimulated by the addition of 1 mM forskolin (FSK) for about 15-30 minutes at about 37xc2x0 C.
The agonist effect of a compound is measured by the simultaneous addition of FSK (1 xcexcM), SRIF-14 (1xe2x88x9212 M to 10xe2x88x926 M) and a test compound (10xe2x88x9210 M to 10xe2x88x925 M).
The antagonist effect of a compound is measured by the simultaneous addition of FSK (1 xcexcM), SRIF-14 (1 to 10 nM) and a test compound (10xe2x88x9210 M to 10xe2x88x925 M).
The reaction medium is removed and 200 ml 0.1 N HCl is added. cAMP is measured using radioimmunoassay method (Kit FlashPlate SMP001A, New England Nuclear).
Radioligand Binding Assay
Membranes for in vitro receptor binding assays were obtained by homogenizing (Polytron, setting 6, 15 sec) the CHO-K1 cells, expressing the hsst receptor subtypes, in ice-cold 50 mM Tris-HCl and centrifuging twice at 39,000 g (10 min), with an intermediate resuspension in fresh buffer. The final pellets were resuspended in 10 mM Tris-HCl for assay. For the hsst1, hsst3, hsst4, hsst5 assays, aliquots of the membrane preparations were incubated (for about 30 min at about 37xc2x0 C. with 0.05 nM [125I-Tyr11]SRIF-14 in 50 mM HEPES (pH 7.4) containing BSA (10 mg/ml); MgCl2 (5 mM)), Trasylol (200 KIU/ml), bacitracin (0.02 mg/ml), and phenylmethylsulphonyl fluoride (0.02 mg/ml). The final assay volume was 0.3 ml.
For the hsst2 assay, [125I]MK-678 (0.05 nM) was employed as the radioligand and the incubation time was about 90 min at about 25xc2x0 C. The incubations were terminated by rapid filtration through GF/C filters (pre-soaked in 0.3% polyethylenimine) using a Brandel filtration manifold. Each tube and filter were then washed three times with 5-ml aliquots of ice-cold buffer.
Specific binding was defined as the total radioligand bound minus that bound in the presence of 1000 nM SRIF-14(hsst1,3,4,5), or 1000 nM MK678 for hsst2.
The compounds of the instant invention can be in vivo assayed for the uses associated with binding to the somatostatin receptor, including specificity binding to the somatostatin subtype receptor(s), according to methods well known to those skilled in the art as exemplified by the following references: I. Shimon, et. al., xe2x80x9cSomatostatin receptor subtype specificity in human fetal pituitary culturesxe2x80x9d, J. Clin. Invest., Vol. 99, No.4, pp. 789-798, 1997; and C. Gilon, et. al., xe2x80x9cA backbone-cyclic, receptor 5-selective somatostatin analogue: Synthesis, bioactivity, and nuclear magnetic resonance conformational analysisxe2x80x9d, J. Med. Chem. 1998, 41, 919-929.
As is well known to those skilled in the art, the known and potential uses of somatostatin agonists and/or antagonists are varied and multitudinous. These varied uses of somatostatin may be summarized as follows:
Somatostatin agonists can be used to suppress growth hormone and more particularly GH secreting adenomas (acromegaly) and TSH secreting adenomas; treat prolactin secreting adenomas; inhibit insulin and/or glucagon and more particularly diabetes mellitus, angiopathy, proliferative retinopathy, dawn phenomenon and nephropathy; inhibition of gastric acid secretion and more particularly peptic ulcers; enterocutaneous and pancreaticocutaneous fistula; irritable bowel syndrome; Dumping syndrome; watery diarrhea syndrome; AIDS related diarrhea; chemotherapy-induced diarrhea; acute or chronic pancreatitis and gastrointestinal hormone secreting tumors; treatment of cancer such as hepatoma; inhibition of angiogenesis; treatment of inflammatory disorders such as arthritis; retinopathy; chronic allograft rejection; angioplasty; preventing graft vessel and gastrointestinal bleeding.
Accordingly, the present invention includes within its scope pharmaceutical compositions comprising, as an active ingredient, at least one of the compounds of the instant invention as described herein in association with a pharmaceutically acceptable carrier.
A compound of this invention can be administered by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous or subcutaneous injection, or implant), nasal, vaginal, rectal, sublingual or topical routes of administration and can be formulated with pharmaceutically acceptable carriers to provide dosage forms appropriate for each route of administration.
Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound is admixed with at least one inert pharmaceutically acceptable carrier such as sucrose, lactose, or starch. Such dosage forms can also comprise, as is normal practice, additional substances other than such inert diluents e.g., lubricating agents such as magnesium stearate. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings.
Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, the elixirs containing inert diluents commonly used in the art, such as water. Besides such inert diluents, compositions can also include adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring and perfuming agents.
Preparations according to this invention for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, or emulsions. Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. Such dosage forms may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. They may be sterilized by, for example, filtration through a bacteria-retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions. They can also be manufactured in the form of sterile solid compositions which can be dissolved in sterile Water, or some other sterile injectable medium immediately before use.
Compositions for rectal or vaginal administration are preferably suppositories which may contain, in addition to the active substance, excipients such as coca butter or a suppository wax.
Compositions for nasal or sublingual administration are also prepared with standard excipients well known in the art.
Further, a compound of this invention can be administered in a sustained release composition such as those described in the following patents. U.S. Pat. No. 5,672,659 teaches sustained release compositions comprising a bioactive agent and a polyester. U.S. Pat. No. 5,595,760 teaches sustained release compositions comprising a bioactive agent in a gelable form. U.S. Pat. No. 5,821,221 teaches polymeric sustained release compositions comprising a bioactive agent and chitosan. U.S. Pat. No. 5,916,883 teaches sustained release compositions comprising a bioactive agent and cyclodextrin. U.S. application Ser. No. 09/015,394 filed Jan. 29, 1998, now abandoned, teaches absorbable sustained release compositions of a bioactive agent. The teachings of the foregoing patents and applications are incorporated herein by reference.
The dosage of active ingredient in the compositions of this invention may be varied; however, it is necessary that the amount of the active ingredient be such that a suitable dosage form is obtained. The selected dosage depends upon the desired therapeutic effect, on the route of administration, and on the duration of the treatment. Generally, dosage levels of between 0.0001 to 100 mg/kg of body weight daily are administered to humans and other animals, e.g., mammals, to obtain a therapeutic effect.
A preferred dosage range is 0.01 to 5.0 mg/kg of body weight daily which can be administered as a single dose or divided into multiple doses.
A compound of this invention can be synthesized according to the following description and Scheme I. In a first step, an amino acid, protected on the xcex1-amino group with Boc, Cbz or other suitable group, is converted to a carboxylate salt with an inorganic base, for example NaOH, KOH, K2CO3, or most preferably Cs2CO3, in a polar solvent such as H2O, DMF, THF, or the like. The solvent is removed under vacuum and the residual salt is re-dissolved in a polar aprotic solvent such as DMF and a suitable xcex1-halo ketone is added with stirring at about xe2x88x9220xc2x0 C. to about 100xc2x0 C., most preferably at room temperature. Stirring is continued for about 10 minutes to about 24 hours, or until ester formation is complete by TLC analysis, at which time the solution is concentrated under vacuum at about 0xc2x0 C. to about 100xc2x0 C., most preferably at about 40xc2x0 C. to about 70xc2x0 C. The intermediate is re-dissolved in an aprotic organic solvent such as benzene, toluene or, most preferably, xylenes, and about 5-fold to about 100-fold or, most preferably about 15-20 fold molar excess of NH4OAc is added. The two phase mixture is heated at reflux and the polar layer is completely removed over the course of about 1 to about 4 hours by means of a Dean-Stark trap to give crude intermediate (A) which may be used crude or purified by crystallization or column chromatography. 
In a second step, intermediate (A) is deprotected using catalytic 5 hydrogenation or strong acids such as HF, HCl, HBr or Tfa. The xcex1-nitrogen may then be protected with a base sensitive protecting group such as the Fmoc group using commercially available N-(9-fluorenylmethoxycarbonyloxy)succinimide and K2CO3 in, for example, acetonitrile and water. Alternatively, the Nxcex1-Cbz-protected imidazole nitrogen may be alkylated with a protected carboxylic ester halide and deprotected on the xcex1-amino group using catalytic hydrogenation to yield Bxe2x80x2 (V=H, W=xe2x80x94(CH2)mCR5CO2Rxe2x80x2, where Rxe2x80x2 represents an alkyl or benzylic ester). The imidazole nitrogen may be protected using commercially available triphenylmethyl chloride and a tertiary amine base such as 4-methyl-morpholine, diisopropylethylamine or triethylamine to yield an Fmoc-protected intermediate which is subsequently deprotected on the xcex1-amino group using bases such as, for example, TAEA to yield intermediate (B) (V=H, W=Trt). Alternatively, the N-deprotected imidazole Bxe2x80x3 (V=H,W=H) may be used without further modification.
In a third step, intermediate B, Bxe2x80x2, or Bxe2x80x3 is used as an anchor group for the continuous solution phase synthesis of a target peptide. Thus, the anchor group is dissolved in ethyl acetate at a concentration of about 50-200 mmol per liter and about 1 to 5 molar equivalents or, more preferably, 1.1 to 1.5 molar equivalents of an Nxcex1-Fmoc protected amino acid, in the form of its activated ester, anhydride or acid halide is added. The mixture is stirred over a second layer of weak base such as aqueous Na2CO3 or, more preferably, aqueous NaHCO3 solution until the reaction is complete. The aqueous layer is removed and about 1 to 10 ml/mmol or, more preferably, about 2-4 ml/mmol of TAEA or piperidine is added and the mixture is stirred for about 30 minutes. The solution is then washed with saturated NaCl solution (2 times with about 30 ml/mmol) and then with 10% phosphate buffer solution adjusted to pH=5.5 (3 times with about 10 ml/mmol). Subsequent cycles are performed in a manner similar to the first cycle. The final amino acid may be protected on Nxcex1 with a Boc or an Fmoc group.
In a fourth step, the N-terminal and C-terminal protecting groups are removed with aqueous base or with strong acids, and the resulting peptide intermediate may be cyclized using classical peptide coupling techniques as described in xe2x80x9cThe Practice of Peptide Synthesisxe2x80x9d, Bodanszky and Bodanszky, Springer-Varlag, 1984. Accordingly, the peptide intermediate is dissolved in an aprotic solvent such as DMF and the solution is made basic by addition of tertiary amine base such as 4-methyl-morpholine. The carboxylate portion of the intermediate is activated by addition of a 1- to 6-fold molar excess of a carbodiimide, such as DCC or EDC, and an additive such as, for example, 1-hydroxybenzotriazole. The mixture is stirred at about 0xc2x0 to 100xc2x0 C., most preferably at about room temperature, until the reaction is complete.
In a final step, the protected peptide is freed of protecting groups using catalytic hydrogenation or strong acids such as HF, HCl, HBr or Tfa to yield final product (C), where R1 to R5, a, b, Y, Z, and n are as defined above for formula (I).
The infusion mass spectral data was measured on a Finnigan SSQ 7000 spectrometer equipped with an ESI (electrospray ionization) source. NMR data was obtained on a 300 MHz Varian Unity spectrometer from samples at concentrations of about 10-20 mg/ml in the designated solvents.
Alternatively, compounds of the present invention can be prepared using solid phase peptide synthesis techniques. Thus, intermediate A (X=Boc) is alkylated with, for example, ethyl bromoacetate and a suitable base, for example, K2CO3 in an aprotic solvent, for example, DMF, and the resulting ethyl ester intermediate is hydrolyzed using an aqueous base, for example, NaOH, to provide intermediate B (V=Boc, W=xe2x80x94CH2CO2H). Intermediate B (V=Boc, W=xe2x80x94CH2CO2H) can be activated using known activation techniques as described in xe2x80x9cThe Practice of Peptide Synthesisxe2x80x9d, Bodanszky and Bodanszky, Springer-Varlag, 1984, and used directly for coupling to the growing peptide on a solid support or, intermediate B (V=Boc, W=xe2x80x94CH2CO2H) may be attached directly to the solid support to begin a solid phase synthesis. Deprotection of the N-terminal Boc group with, for example, Tfa, allows the continuation of peptide synthesis under conditions known to one of ordinary skill in the art.
Intermediate B (V=Fmoc, W=xe2x80x94CH2CO2t-Bu, for example) can be treated with acid, for instance, Tfa to remove the carboxylate protecting t-butyl ester and the resulting intermediate B (V=Fmoc, W=xe2x80x94CH2CO2H, for example) can be used for solid phase peptide synthesis using the Fmoc strategy. Thus, Intermediate B (V=Fmoc, W=xe2x80x94CH2CO2H) can be activated using known activation techniques as described in xe2x80x9cThe Practice of Peptide Synthesisxe2x80x9d, M. Bodanszky and A. Bodanszky, Springer-Varlag, 1984, and used directly for coupling to the growing peptide on a solid support. Deprotection of the N-terminal Fmoc group with, for instance, piperidine allows the continuation of peptide synthesis under conditions known to one of ordinary skill in the art.
The solid phase synthesis of cyclic analogs can also be carried out, according to Scheme II, below. 
Intermediate A (X=Boc or Cbz, R3=2-methoxyphenyl, 3-methoxyphenyl or 4-methoxyphenyl) can be treated with 1M BBr3 in CH2Cl2 for about xc2xd hour to provide the free phenol A (X=H, R3=2-hydroxyphenyl, 3-hydroxyphenyl or 4-hydroxyphenyl). The nitrogen may then be protected with an acid sensitive protecting group such as the Boc group using di-t-butyldicarbonate and a base, for example, NaOH in a mixture of an organic, water miscible solvent, for example, dioxane and water. Intermediate A (X=Boc, R3=2-hydroxyphenyl, 3-hydroxyphenyl or 4-hydroxyphenyl) is alkylated with, for example, ethyl bromoacetate and a suitable base, for example, K2CO3 in an aprotic solvent, for example, DMF, to provide the resulting ethyl ester intermediate D. Intermediate D is then converted to it""s cesium salt by the action of cesium carbonate. The cesium salt is reacted, in excess, with Merrifield resin to provide intermediate E. Intermediate E is subject to elaboration using standard Boc solid phase peptide synthesis or standard Fmoc solid phase synthesis as previously described to yield intermediate F. When the complete amino acid sequence has been constructed, the C-terminal ethyl ester is unmasked using a suitable base, for example, LiOH in aqueous DMF and the peptide is cyclized using standard activation protocol, for example, a carbodiimide with, for example, hydroxybenzotriazole and a tertiary amine base, for example, diisopropylethyl amine to provide intermediate G. Final side chain deprotection and cleavage from the resin is realized by addition of a very strong acid, for example, HF to yield compounds of the invention.